With the support of the current funded R01, we have modified and optimized our nanovectors for tumor targeted delivery of siRNA/shRNA specific to Bcl-2 and Bcl-xL. We found unexpectedly that our nanovectors also targeted to pancreatic cancer stem cells (CSC) and delivered siRNA/shRNA to CSC, inhibited CSC selfrenewal and reduced cancer stem cells in vivo. Based on our exciting new discovery, we propose, in this Competitive Revision project, to expand the scope of the current R01 to further develop the nanovectors for delivery of miRNA-therapeutics targeting cancer stem cells. This is in response to the NOT-OD-09-058: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. Our long-term goal is to develop nanovector-miRNA as a novel molecular therapy targeting human cancer stem cells. The objective in this two-year project is to explore our above patented nanovector system for targeted delivery of miRNA-based therapeutics to human pancreatic cancer stem cells, and to evaluate the therapeutic potential of the nanovector-delivered tumor suppressor miR-34 in inhibiting CSC. We will test two inter-related basic hypotheses: (1) the self-assembled nanovectors can efficiently deliver miR-34 to the pancreatic cancer stem cells, thus restoring the miR-34 tumor suppressor function in cancer stem cells;(2) miR-34 restoration will inhibit the self-renewal of pancreatic cancer stem cells by simultaneously inhibiting Bcl-2 and Notch, and thus provide a potential novel therapy for pancreatic cancer. To test our hypothesis, we propose to carry out two SPECIFIC AIMS: AIM 1, To prepare and characterize the miRNA-nanovectors targeting CSC in vitro and in vivo;AIM 2, To investigate the anti-tumor activities and the mechanism of action of nanovector-miR-34. Our long-term goal is to develop nanovector-miRNA as a novel molecular therapy targeting human cancer stem cells. Successfully carried out, our studies will provide novel, self-assembled miRNA-nanovectors that can deliver tumor suppressor miR-34 to pancreatic CSC and inhibit their self-renewal and tumor-initiation. The tumor specificity of nanovector-miR-34 is based upon: (1) cancer stem cells have high level of TfR and dysregulation of miR-34-Notch/Bcl-2 for excessive self-renewal;(2) the optimized formulation of nanovectors based on preferred miR-34 delivery to and functional inhibition of CSC;(3) normal stem cells, although also TfR(+), have no dysregulation of Bcl-2/Notch and do not rely on Bcl-2/Notch for survival. The data from the current proposal will provide important impetus to develop the nanovector-miR-34 as a novel and more effective therapy for human pancreatic cancer, potentially by modulating pancreatic cancer stem cells. PUBLIC HEALTH RELAVANCE: Our objective is to explore our patented self-assembled nanovector system for targeted delivery of miRNA-based therapeutics to human pancreatic cancer stem cells, and to evaluate the therapeutic potential of the nanovector-delivered tumor suppressor miR-34 in inhibiting cancer stem cells. Successfully carried out, our studies will provide novel, self-assembled miRNA-nanovectors that can deliver tumor suppressor miR-34 to pancreatic cancer stem cells and inhibit their self-renewal and tumor-initiation. The data from the current proposal will provide important impetus to develop the nanovector-miR-34 as a novel and more effective therapy for human pancreatic cancer by targeting cancer stem cells.